Abstract: In order to compare and analyze the differences of fungi in different cultivated soils/substrates of pepper and reveal the characteristics of fungal community structure in the soilless culture substrates of pepper, the rhizosphere matrix (SlPlRh) for the soilless culture of pepper, non-rhizosphere matrix for the soilless culture (SlPlNRh), rhizosphere soil (PlRh), non-rhizosphere soil (PlNRh) and uncultivated soil (NPl) were collected as the experimental materials. Then, the diversity and abundance of fungi in the five kinds of soils/substrates were detected by using the Illumina Seq high-throughput sequencing technology, and the structural characteristics of fungal community were analyzed. The results showed that PlNRh had the highest fungal abundance and diversity, while SlPlNRh had the lowest. And there were significant differences in the abundance between the two samples, while there were no significant differences in the abundance and diversity among other samples. The fungal community composition of PlRh, SlPlRh and SlPlNRh was similar at the phylum classification level, and the average relative abundance ratio was different. At the genus classification level, the composition of NPl, PlRh and PlNRh was similar, and the composition of SlPlRh and SlPlNRh was similar. The cluster analysis of fungal community showed that the three kinds of soil (NPl, PlRh and PlNRh) and the two kinds of substrates (SlPlRh and SlPlNRh) were obviously divided into two class groups. The lEfSe analysis showed that PlRh and PlNRh had the most species with significant difference, while SlPlRh had the fewest. By detecting the diversity and community composition of fungi in the planting soil and the soilless culture substrates of pepper, the differences of the rhizosphere fungal microbiome in different types of planting substrates were compared and analyzed. Then, the diversity and community structure characteristics of fungi in the soilless culture substrates of pepper were preliminarily revealed, which provided scientific basis for the selection of soilless culture substrates, the distribution ratio and improvement of matrix group in the future.